1. Field of the Invention
The present invention relates to a novel analytical method and device for analyzing cells, DNA, antigens and viruses in the field of laboratory tests such as a clinical examination. In particular, the invention relates to a novel, analytical method and device to combine cytometry and cytodiagnosis.
2. Related Background Art
A flow cytometer is a popular analytical device for flow cytometry. Flow cytometry is an analytical method where a cellular surface antigen is stained with one or more fluorescence-labeled specific antibodies, or DNA is stained with various dyes that bind to DNA, and obtained parameters, i.e., intensities of fluorescence, respective intensities of forward and side-way scattering light, and the individual numbers, are subjected to various analyses. On the other hand, in the fields of pathology, cytodiagnosis and hemodiagnosis, usually chemically stained smears are prepared and judged on a microscopic level.
In clinical cytodiagnosis, final judgment by an expert is still required in many cases, no matter how instruments have been automated and techniques for judging with image processing have been advanced. On the other hand, in flow cytometry, image observation is not involved. Thus, although flow cytometry has been recognized as a superior clinical test method because of its operability and simplicity, valuable information may be wasted. Under such circumstances, Japanese Patent Application Laid-Open No. 5-119035 (xe2x80x9cImaging Flow Cytometerxe2x80x9d) disclosed a flow cytometer having an image-pick up function. However, this device cannot provide specimens, because cells flow out from the flow cell.
Accordingly, an object of the invention is to provide a novel analytical device that can function to both prepare a specimen required for current cytodiagnosis and as a flow cytometer, and provide a novel, analytical method of flow cytometry with specimen preparation.
In order to overcome the above-described problem, the inventor conducted an intensive investigation and has finally invented a novel, analytical method and device, which have both an imaging function and specimen-preparation function by combining a centrifugation method and a fluorescence-labeling technique, such as flow cytometry. This analytical device is, so to speak, a flow cytometer having imaging and specimen-preparing functions. Here, this analytical device is referred to as a disc cytometer, because it typically uses a disc-shaped sample container, and the method is referred to as flow cytometry. It should be understood, however, that the shape of the sample container is not limited to a disc, as will be evident from the following description. The sample container must be made up of an optically transparent material, because it is used for cytodiagnosis, immunofluorescence analysis, images of cells etc.
This disc cytometry adopts differential centrifugation or density-gradient centrifugation, which has been used for isolating and purifying biological materials. In density-gradient centrifugation, a sample in a centrifuge tube is spun, and substances in the sample are distributed along a density gradient according to the differences in size, shape, density or specific gravity. In this invention, a plate-shaped sample container is used, which enables in situ cytodiagnosis as well as analysis and imaging by the fluorescence-labeling method in one container, instead of a cylindrical centrifuging tube employed in conventional density-gradient centrifugation. The sample container is preferably in the form of a disc in view of centrifugation, but it can be in any form as long as it is suitable for centrifugation, for example, a rectangular sample container.
As the medium for generating a density gradient during centrifugation, PEG (polyethylene glycol) or sucrose in an appropriate range is used for biological samples. In clinical laboratory tests, commercial solutions such as Ficoll (trade name) and Percoll (trade name) may be used, depending on the purpose.
One aspect of the present invention is an analytical method comprising the steps of: preparing a sample containing particulate substances; injecting the sample into a plate-like sample container; centrifuging the sample container; and using the sample container in which a distribution of the particulate substances has been formed as a preparation for analysis.
The term xe2x80x9cparticulate substancesxe2x80x9d, xe2x80x9ccellsxe2x80x9d, etc. as used herein refer to biological materials, which are subjected to cytodiagnosis or clinical laboratory test, for example, those selected from the group consisting of cells, microbial cells, viruses, DNA and mitochondria.
Further, in this invention, the sample container preparation is irradiated and scanned with a laser beam to obtain at least one set of analytical data selected from fluorescence intensity, scattered light intensity and light scattering particle number for particulate substances in the sample. The particulate substances may be subjected to a fluorescence labeling reaction before centrifugation. The image data of particulate substances may be obtained on the basis of analytical data.
In this invention, the same experimental protocols as used in conventional flow cytometry can be used for sample preparation and reaction with florescent antibodies. Accordingly, when a suitably reacted sample is introduced and overlayed on the density gradient solution in a disc container, and centrifuged for a predetermined time at a predetermined rotation speed, the cells, etc. are arranged along the formed density gradient. Then, a laser scan of the disc container is carried out in the radius direction with rotation of the container to obtain information such as fluorescence intensity, scattered light intensity and the individual number. Depending on the purpose, image information at a certain position of the disc container can be obtained by using positional information of the disc container from a sensor and an optical system of confocal laser microscope.
This is the disc-cytometry analysis of the present invention.
Another aspect of the present invention is an analytical device comprising: means for centrifuging a plate-like sample container; means for generating a laser beam; means for scanning the sample container and irradiating particulate substances having been centrifuged in the sample container with a laser beam; and means for detecting scattered light from the sample container.
In this invention, the means for detecting scattered light may be a means for detecting light scattered from the disc container at different angles. In such a case, preferably, the fluorescence intensities and/or the number of the particulate substances are detected based on the a plurality of scattered light. Preferably, the irradiating means scans the rotating disc container in a radius direction with a laser beam.
Further, the analytical device of this invention may be provided with a means for processing and analyzing data by A/D converting the signals from the scattered light detecting means and using the same as a parameter. In addition, it may be provided with an optical system and an image pick-up means for obtaining images of the particulate substances.
The specimen-providing function of the present invention is very useful for clinical judgement as a supplementary confirmation means. For example, it can be used for discrimination of leukemia from malignant lymphoma, acute lymphoblastic leukemia from acute crisis of chronic myelogenous leukemia, and lymphoblastic leukemia from nonlymphoblastic leukemia, all of which are considered to be difficult to discriminate only by current flow cytometry. Also, it can be used for identification of immature T cells and for identification of neoplasm, which is difficult to identify solely by the DNA Index.